Journal: bioRxiv

Article Title: Brainstem enkephalinergic neural circuit underlying cold-induced pain relief in mice

doi: 10.1101/2025.11.10.686886

Figure Lengend Snippet: (A) Experimental scheme for preparation of single-cell suspension and sorting of Zsg-positive neuron of lPBN. (B) Percentage of Zsg-positive cells in total viable cells in Wildtype mice and TRAP2 x Ai6 mice. (C) Percentage of viable cells in total counts. (D) Gating strategy for fluorescence-based sorting of Zsg-positive, viable neuron from brain single cell suspension. (E) Volcano plot of significantly enriched genes (Red dot) and depleted genes (Blue dot) in Zsg-positive cells compared to Zsg-negative cells. (F) Representative figure of RNAscope fluorescence in situ hybridization in lPBN (left) and quantification of marker co-expression in Zsg-expressing lPBN neurons (right). Red; Penk, Green; Zsgreen, Blue; DAPI (left) Scale bar: 20μm. (G) Experimental scheme for the selective activation of lPBN cold neurons for enkephalin release measurement. (H) Representative figure of met-enkephalin immunostaining in vlPAG, with lPBN cold neurons activity manipulation (top) and enlarged images from ventrolateral subcolumn (vl) and dorsolateral subcolumn (dl). Green; Met-enkephalin. Scale bar: 100μm. (I) Quantification of met-enkephalin level in vlPAG after lPBN cold neuron activity manipulation. (J) Quantification of met-enkephalin level in vlPAG, in control mice and cold-exposed mice. (K) Experimental scheme for the local neutralizing antibody treatment in vlPAG before TRPM8-mediated analgesia behavioral tests. (L) Mechanical sensitivity measured with 50% withdrawal thresholds in CFA-induced inflammatory pain model, with neutralizing antibody administration in the vlPAG. Data are presented as mean ± SEMs. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001. Number of mice and statistical tests are listed in Table S1 .

Article Snippet: RNAscope multiplex v2 kit (Advanced Cell Diagnostics (ACD)) was used to perform fluorescence in situ hybridization according to the manufacturer’s protocols.

Techniques: Single Cell, Suspension, Fluorescence, RNAscope, In Situ Hybridization, Marker, Expressing, Activation Assay, Immunostaining, Activity Assay, Control